The present invention provides chemokine-like factors (CKLFs) polypeptides with chemotactic and hematopoietic stimulating activities and polynucleotides encoding such polypeptides. Also provided is the method to produce such polynucleotides and polypeptides. The present invention further discloses a drug compound comprising a therapeutically effective amount of such CKLF polypeptides as well as pharmaceutically acceptable excipients and carriers. Also disclosed are the uses of such CKLF polypeptides and polynucleotides in diagnosis or treatment of immunodeficiency, hematopoietic diseases and primary tumors. The present invention also relates to the antibodies and antagonists of such CKLF polynucleotides.
Cytokines are a superfamily of small proteins synthesized and secreted by different cells, which participate in the proliferation and differentiation of many kinds of cells and play essential roles in physiological and pathological processes. Cytokines include interleukins, colony stimulating factors, interferons, tumor necrosis factors, growth factors and chemokines. Many kinds of recombinant cytokines and their antagonists produced by using genetic engineering technology have been applied to clinical treatment and have enjoyed positive effects. This reflects the spacious applications of cytokines.
The superfamily of chemokine (also called chemotactic factor) comprises dozens of polypeptides with molecular weights of 8-12 KD. These polypeptides are similar in structure and in their chemotactic effects, and they play important roles in immune defense, immunoregulation, inflammation, hematopoietic regulation and vasculogenesis. The amino acid sequences of most of the chemokines share the same character of conserved four-cysteine (Cys) motif. According to the positions of the first and the second Cys, chemokines can be divided into four subfamilies named CXC, CC, CX3C and C, respectively, wherein C represents Cys and X represents any kind of amino acid. The members of CXC, such as IL-8, interferon-induced protein 10 (IFN-IP-10), and cytokine with melanocyte growth stimulating activity (MGSA) have their encoding genes usually located on chromosome 4 in human somatic cells, and activate and attract neutrophils and T lymphocytes. While the numbers of CC, such as macrophage inflammatory protein-1xcex1 (MIP-1xcex1), macrophage inflammatory protein-1xcex2(MIP-1xcex2), monocyte chemoattractant protein-1(MCP-1) and cytokine regulated on activation, and normal T cell expressed and secreted (RANTES) have their encoding genes usually located on chromosome 17 in somatic cells, and mainly activate monocytes, lymphocytes, basophils and eosinophils. Fractalkine is the only identified member of the CX3C subfamily, which can activate and chemotacte T cells and monocytes. As for the C subfamily, only Lymphotactin (Ltn) is presently found, which can chemotacte lymphocytes.
The receptors of chemokines belong to the superfamily of GTP-binding protein which have the characteristic of seven transmembrane domains. In accordance with the specific combination to different numbers of chemokine subfamilies, chemokine receptors are named: CXC Receptor (CXCR), such as CXCR 1, CXCR 2, CXCR 3, and CXCR 4; CC Receptor (CCR), such as CCR 1, CCR 2, CCR 3, CCR 4, CCR 5, CCR 6 and CCR 7; CX3C Receptor (CX3CR) (See Marco, B., et al., 1997, Human Chemokines: An Update Annu. Rev. Immunol. 15:675-705). Because chemokines play important roles in system immunization, inflammatory responses and hematopoietic regulation, some abnormal changes of chemokines or their receptors (e.g., the defect of chemokines or chemokine receptors, or the overexpression of chemokines, or the increase of soluble chemokine receptors) always result in some kind of infectious disease or autoimmune disease, or sometimes even in tumors. Therefore, the detection of chemokines or chemokine receptors can be used in clinical diagnosis to observe the development of diseases and to judge the curative effects. Recent studies show that HIV can invade the immune cells of human by combining with chemokine receptors, which indicate that chemokine receptors as well as chemokines are closely related to the infection and development of HIV disease. Hence, Chemokines could be used as promising drugs to block the invasion of HIV to immune cells (Cocchi, F., et al., 1995, xe2x80x9cIdentification of RANTES, MIP-1xcex1, and MIP-1xcex2 as the Major HIV-Suppressive Factors Produced by CD8+ T Cells,xe2x80x9d Science 270:1811). Furthermore, some chemokine drugs produced by bioengineering methods have been successively used in clinical tests and have the possibility to become useful medicines in the future. For instance, myeloid progenitor inhibitory factor-1 (MPIF-1) has been used as a protective hematopoietic drug in high-dose radiotherapy or chemotherapy for tumors (Marshall, A., 1998, xe2x80x9cHGS launches xe2x80x9cfirstxe2x80x9d genomics product in clinic,xe2x80x9d Nat. Biotechol. 16:129).
The present invention is based on the finding that many types of cytokines or chemokines can be stimulated by phytohemagglutinin (PHA) (Brantschen, S., et al., 1989, xe2x80x9cDifferential expression of cytokine mRNAs cell lines,xe2x80x9d Lymphokine Res 8(3):163-72), while interleukin-10(IL-10) is a broad-spectrum inhibitor of cytokines (Di-Hwei, H., et al., 1990, Science 250:830). By using suppression subtractive hybridization (SSH) technique, some novel cytokines could be found. (Diatchenko, L., et al, 1996, xe2x80x9cSuppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries,xe2x80x9d Pro. Natl Acad. Sci. USA 93:6025-30). Based on the above, the present inventors, by using SSH technique, identified and cloned CKLF1 gene from a cDNA library derived from U937 cell line, a human promonocytic cell line. Furthermore, the inventors disclose the biological activity and function of CKLF1 and its variants.
One aspect of the present invention relates to novel mature CKLF polypeptides which have chemotactic activities on immune cells and stimulatory effects on hematopoietic progenitor cells. Also provided are biologically active and diagnostically or therapeutically useful fragments, variants, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.
Another aspect of the present invention relates to isolated nucleic acid molecules encoding polypeptides of the present invention, including mRNAs, cDNAs, genomic DNAs as well as variants, analogs, derivatives and biologically active and diagnostically or therapeutically useful fragments thereof.
Another aspect of the present invention relates to a method for producing such CKLF polypeptides by recombinant techniques comprising cloning nucleic acid sequences encoding the polypeptides of the present invention into suitable vectors, introducing recombinant vectors into prokaryotic and/or eukaryotic host cells by transduction, transfection or transformation methods, and culturing such prokaryotic and/or eukaryotic host cells under suitable conditions to express the CKLF polypeptides of the present invention.
Another aspect of the present invention relates to diagnostic or therapeutic uses of the CKLF polypeptides and polynucleotides in the treatment of immunodeficiency, hematopoietic diseases and primary tumors. It further relates to a method for diagnosing a disease or susceptibility to a disease comprising determining a mutation in the polynucleotide of the present invention. It still further relates to a method of diagnosis comprising analyzing for the presence of the polypeptide of the present invention.
Another aspect of the present invention relates to drug compounds comprising a therapeutically effective amount of such CKLF polypeptides or its functional fragments as well as pharmaceutically acceptable excipients and carriers. It further relates to a method for the treatment of a patient having need of CKLF comprising administering to said patient a therapeutically effective amount of the polypeptide.
Still another aspect of the present invention relates to polyclonal or monoclonal antibodies of the polypeptides of the present invention. It further relates to a method of using the antibody in the preparation of immunological adjuvants which can improve the curative effect of a DNA vaccine or a DNA drug.
Yet another aspect of the present invention relates to antagonists of the polypeptides of the present invention, which may be used to inhibit the action of such polypeptides, for example, in the treatment of rheumatoid arthritis, autoimmune diseases, tumors, and viral infections.